publications

2025

1. Coordination of Focal Adhesion Nanoarchitecture and Dynamics in Mechanosensing for Cardiomyoblast Differentiation

   J. Xiao ,  J. W. Ang ,  X. Zhong ,  D. C. P. Wong ,  T. T ,  I. Yow ,  C. J. M. Lee ,  R. S. Foo ,  P. Kanchanawong ,  and  B. C. Low

   2025

   Xiao, Jingwei Ang, Jing Wen Zhong, Xueying Wong, Darren Chen Pei T, Thivakar Yow, Ivan Lee, Chang Jie Mick Foo, Roger S-Y Kanchanawong, Pakorn Low, Boon Chuan eng 2025/01/09 ACS Appl Mater Interfaces. 2025 Jan 22;17(3):4463-4479. doi: 10.1021/acsami.4c15459. Epub 2025 Jan 8.

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   Focal adhesions (FAs) are force-bearing multiprotein complexes, whose nanoscale organization and signaling are essential for cell growth and differentiation. However, the specific organization of FA components to exert spatiotemporal activation of FA proteins for force sensing and transduction remains unclear. In this study, we unveil the intricacies of FA protein nanoarchitecture and that its dynamics are coordinated by a molecular scaffold protein, BNIP-2, to initiate downstream signal transduction for cardiomyoblast differentiation. Within the FAs, BNIP-2 regulates the nano-organization of focal adhesion kinase (FAK), and the dynamics of FAK, paxillin, and vinculin. Depletion of BNIP-2 resulted in altered focal adhesion numbers and sizes per cell, reduced traction force, and decreased FA sensitivity for mechanosensing. At the molecular level, the loss of BNIP-2 disrupted the FAK-paxillin signaling axis, where FAK inhibition reproduces the effects of BNIP-2 loss by impairing the phosphorylation of both FAK and paxillin. Mechanistically, BNIP-2 preferentially binds to constitutively active FAK and acts as a molecular scaffold to mediate interactions between FAK and paxillin and between paxillin and vinculin. We have validated BNIP-2’s role in the FAK-paxillin signaling axis in human embryonic stem cells (hESC). Furthermore, we showed that depletion of BNIP-2 resulted in changes in signature gene targets at the cardiac progenitor stage of differentiation. In summary, we showed that the intricate interplay of FA nanoarchitecture and dynamics, governed by BNIP-2, is crucial for force transduction and biochemical signaling in driving cardiomyoblast differentiation.

2. The epigenetic basis of hepatocellular carcinoma - mechanisms and potential directions for biomarkers and therapeutics

   H. Y. Lin ,  A. J. Jeon ,  K. Chen ,  C. J. M. Lee ,  L. Wu ,  S. L. Chong ,  C. G. Anene-Nzelu ,  R. S. Foo ,  and  P. K. Chow

   2025

   Lin, Hong-Yi Jeon, Ah-Jung Chen, Kaina Lee, Chang Jie Mick Wu, Lingyan Chong, Shay-Lee Anene-Nzelu, Chukwuemeka George Foo, Roger Sik-Yin Chow, Pierce Kah-Hoe eng Review England 2025/03/09 Br J Cancer. 2025 Mar 8. doi: 10.1038/s41416-025-02969-8.

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   Hepatocellular carcinoma (HCC) is the sixth leading cancer worldwide and has complex pathogenesis due to its heterogeneity, along with poor prognoses. Diagnosis is often late as current screening methods have limited sensitivity for early HCC. Moreover, current treatment regimens for intermediate-to-advanced HCC have high resistance rates, no robust predictive biomarkers, and limited survival benefits. A deeper understanding of the molecular biology of HCC may enhance tumor characterization and targeting of key carcinogenic signatures. The epigenetic landscape of HCC includes complex hallmarks of 1) global DNA hypomethylation of oncogenes and hypermethylation of tumor suppressors; 2) histone modifications, altering chromatin accessibility to upregulate oncogene expression, and/or suppress tumor suppressor gene expression; 3) genome-wide rearrangement of chromatin loops facilitating distal enhancer-promoter oncogenic interactions; and 4) RNA regulation via translational repression by microRNAs (miRNAs) and RNA modifications. Additionally, it is useful to consider etiology-specific epigenetic aberrancies, especially in viral hepatitis and metabolic dysfunction-associated steatotic liver disease (MASLD), which are the main risk factors of HCC. This article comprehensively explores the epigenetic signatures in HCC, highlighting their potential as biomarkers and therapeutic targets. Additionally, we examine how etiology-specific epigenetic patterns and the integration of epigenetic therapies with immunotherapy could advance personalized HCC treatment strategies.

3. Cardiovascular, Kidney, Liver, and Metabolic Interactions in Heart Failure: Breaking Down Silos

   C. J. M. Lee ,  L. B. Kosyakovsky ,  M. S. Khan ,  F. Wu ,  G. Chen ,  J. A. Hill ,  J. E. Ho ,  R. S. Foo ,  and  F. Zannad

   2025

   Lee, Chang Jie Mick Kosyakovsky, Leah B Khan, Muhammad Shahzeb Wu, Feng Chen, Guo Hill, Joseph A Ho, Jennifer E Foo, Roger S-Y Zannad, Faiez eng Review 2025/05/22 Circ Res. 2025 May 23;136(11):1170-1207. doi: 10.1161/CIRCRESAHA.125.325602. Epub 2025 May 22.

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   Over the past few decades, the rising burden of metabolic disease, including type 2 diabetes, prediabetes, obesity, and metabolic dysfunction-associated steatotic liver disease, has corresponded with fundamental shifts in the landscape of heart failure (HF) epidemiology, including the rising prevalence of HF with preserved ejection fraction. It has become increasingly important to understand the role of extracardiac contributors and interorgan communication in the pathophysiology and phenotypic heterogeneity of HF. Whereas traditional epidemiological strategies have separately examined individual contributions of specific comorbidities to HF risk, these approaches may not capture the shared mechanisms and more complex, bidirectional relationships between cardiac and noncardiac comorbidities. In this review, we highlight the cardiac, kidney, liver, and metabolism multiorgan interactions and pathways that complicate HF development and progression and propose research strategies to further understand HF in the context of multiple organ disease. This includes evolving epidemiological approaches such as multiomics and machine learning which may better capture common underlying mechanisms and interorgan crosstalk. We review existing preclinical models of HF and how they have enhanced our understanding of the role of multiorgan disease in the development of HF subtypes. We suggest recommendations as to how clinical practice across multiple specialties should screen for and manage multiorgan involvement in HF. Finally, recognizing the advent of novel combinatorial therapeutic agents that may have multiple indications across the cardiac-kidney-liver metabolism continuum, we review the current clinical trials landscape. We specifically highlight a pressing need for the design of more inclusive trials that examine the contributions of multimorbidity and incorporate multiorgan end points, which we propose may lead to outcomes that are evermore clinically relevant today.

4. Metabolome-wide association identifies ferredoxin-1 (FDX1) as a determinant of cholesterol metabolism and cardiovascular risk in Asian populations

   N. Sadhu ,  R. Dalan ,  P. R. Jain ,  C. J. M. Lee ,  L. S. Pakkiri ,  K. Y. Tay ,  T. H. Mina ,  D. Low ,  Y. Min ,  M. Ackers-Johnson ,  T. T. Thi ,  V. G. Kota ,  Y. Shi ,  Y. Liu ,  H. Yu ,  V. Lai ,  Y. Yang ,  D. Tay ,  H. K. Ng ,  X. Wang ,  K. E. Wong ,  M. Lam ,  X. L. Guan ,  N. Bertin ,  E. Wong ,  J. Best ,  R. Sarangarajan ,  P. Elliott ,  E. Riboli ,  J. Lee ,  E. S. Lee ,  J. Ngeow ,  P. Tan ,  C. Cheung ,  C. L. Drum ,  R. S. Foo ,  G. A. Michelotti ,  H. Yu ,  P. A. Sheridan ,  M. Loh ,  and  J. C. Chambers

   2025

   Sadhu, Nilanjana Dalan, Rinkoo Jain, Pritesh R Lee, Chang Jie Mick Pakkiri, Leroy Sivappiragasam Tay, Kai Yi Mina, Theresia H Low, Dorrain Min, Yilin Ackers-Johnson, Matthew Thi, Thi Tun Kota, Vishnu Goutham Shi, Yu Liu, Yan Yu, Hanry Lai, Vicky Yang, Yang Tay, Darwin Ng, Hong Kiat Wang, Xiaoyan Wong, Kari E Lam, Max Guan, Xue Li Bertin, Nicolas Wong, Eleanor Best, James Sarangarajan, Rangaprasad Elliott, Paul Riboli, Elio Lee, Jimmy Lee, Eng Sing Ngeow, Joanne Tan, Patrick Cheung, Christine Drum, Chester Lee Foo, Roger Sy Michelotti, Gregory A Yu, Haojie Sheridan, Patricia A Loh, Marie Chambers, John C eng NMRC/StaR/0028/2017/MOH | National Medical Research Council (NMRC)/ MOH-000271/MOH | National Medical Research Council (NMRC)/ NMRC/PRECISE/2020/MOH | National Medical Research Council (NMRC)/ MOH-001712-01/MOH | National Medical Research Council (NMRC)/ CS-IRG - MOH-001480-00/MOH | National Medical Research Council (NMRC)/ MOH-000896-00/MOH | National Medical Research Council (NMRC)/ H17/01/a0/007/Agency for Science, Technology and Research (A*STAR)/ H18/01/a0/016/Agency for Science, Technology and Research (A*STAR)/ MOE-T2EP30122-0018/Ministry of Education - Singapore (MOE)/ MOH-000333-00/Ministry of Education - Singapore (MOE)/ MOE-000322-00/Ministry of Education - Singapore (MOE)/ MOH-001226-00/Ministry of Health -Singapore (MOH)/ England 2025/05/14 Nat Cardiovasc Res. 2025 May;4(5):567-583. doi: 10.1038/s44161-025-00638-w. Epub 2025 May 13.

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   The burden of cardiovascular disease is rising in the Asia-Pacific region, in contrast to falling cardiovascular disease mortality rates in Europe and North America. Here we perform quantification of 883 metabolites by untargeted mass spectroscopy in 8,124 Asian adults and investigate their relationships with carotid intima media thickness, a marker of atherosclerosis. Plasma concentrations of 3beta-hydroxy-5-cholestenoate (3BH5C), a cholesterol metabolite, were inversely associated with carotid intima media thickness, and Mendelian randomization studies supported a causal relationship between 3BH5C and coronary artery disease. The observed effect size was 5- to 6-fold higher in Asians than Europeans. Colocalization analyses indicated the presence of a shared causal variant between 3BH5C plasma levels and messenger RNA and protein expression of ferredoxin-1 (FDX1), a protein that is essential for sterol and bile acid synthesis. We validated FDX1 as a regulator of 3BH5C synthesis in hepatocytes and macrophages and demonstrated its role in cholesterol efflux in macrophages and aortic smooth muscle cells, using knockout and overexpression models.

2024

1. Computationally defined and in vitro validated putative genomic safe harbour loci for transgene expression in human cells

   M. I. Autio ,  E. Motakis ,  A. Perrin ,  T. Bin Amin ,  Z. Tiang ,  D. V. Do ,  J. Wang ,  J. Tan ,  S. S. L. Ding ,  W. X. Tan ,  C. J. M. Lee ,  A. K. K. Teo ,  and  R. S. Y. Foo

   2024

   Autio, Matias I Motakis, Efthymios Perrin, Arnaud Bin Amin, Talal Tiang, Zenia Do, Dang Vinh Wang, Jiaxu Tan, Joanna Ding, Shirley Suet Lee Tan, Wei Xuan Lee, Chang Jie Mick Teo, Adrian Kee Keong Foo, Roger S Y eng 1610851033/Biomedical Research Council/ 202D8020/Agency for Science, Technology and Research/ England 2024/01/02 Elife. 2024 Jan 2;13:e79592. doi: 10.7554/eLife.79592.

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   Selection of the target site is an inherent question for any project aiming for directed transgene integration. Genomic safe harbour (GSH) loci have been proposed as safe sites in the human genome for transgene integration. Although several sites have been characterised for transgene integration in the literature, most of these do not meet criteria set out for a GSH and the limited set that do have not been characterised extensively. Here, we conducted a computational analysis using publicly available data to identify 25 unique putative GSH loci that reside in active chromosomal compartments. We validated stable transgene expression and minimal disruption of the native transcriptome in three GSH sites in vitro using human embryonic stem cells (hESCs) and their differentiated progeny. Furthermore, for easy targeted transgene expression, we have engineered constitutive landing pad expression constructs into the three validated GSH in hESCs.

2. Genome-Wide CRISPR Screen Identifies an NF2-Adherens Junction Mechanistic Dependency for Cardiac Lineage

   C. J. M. Lee ,  M. I. Autio ,  W. H. Zheng ,  Y. Song ,  S. C. Wang ,  D. C. P. Wong ,  J. Xiao ,  Y. Zhu ,  P. Yusoff ,  X. Yei ,  W. K. Chock ,  B. C. Low ,  M. Sudol ,  and  R. S. Foo

   2024

   Lee, Chang Jie Mick Autio, Matias I Zheng, Wen Hao Song, Yoohyun Wang, Shyi Chyi Wong, Darren Chen Pei Xiao, Jingwei Zhu, Yike Yusoff, Permeen Yei, Xi Chock, Wan Kee Low, Boon Chuan Sudol, Marius Foo, Roger S-Y eng 2024/05/16 Circulation. 2024 May 16. doi: 10.1161/CIRCULATIONAHA.122.061335.

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   BACKGROUND: Cardiomyocyte differentiation involves a stepwise clearance of repressors and fate-restricting regulators through the modulation of BMP (bone morphogenic protein)/Wnt-signaling pathways. However, the mechanisms and how regulatory roadblocks are removed with specific developmental signaling pathways remain unclear. METHODS: We conducted a genome-wide CRISPR screen to uncover essential regulators of cardiomyocyte specification in human embryonic stem cells using a myosin heavy chain 6 (MYH6)-GFP (green fluorescence protein) reporter system. After an independent secondary sgRNA validation of 25 candidates, we identified NF2 (neurofibromin 2), a moesin-ezrin-radixin like (MERLIN) tumor suppressor, as an upstream driver of early cardiomyocyte lineage specification. Independent monoclonal NF2 knockouts were generated using CRISPR-Cas9, and cell states were inferred through bulk RNA sequencing and protein expression analysis across differentiation time points. Terminal lineage differentiation was assessed by using an in vitro 2-dimensional-micropatterned gastruloid model, trilineage differentiation, and cardiomyocyte differentiation. Protein interaction and post-translation modification of NF2 with its interacting partners were assessed using site-directed mutagenesis, coimmunoprecipitation, and proximity ligation assays. RESULTS: Transcriptional regulation and trajectory inference from NF2-null cells reveal the loss of cardiomyocyte identity and the acquisition of nonmesodermal identity. Sustained elevation of early mesoderm lineage repressor SOX2 and upregulation of late anticardiac regulators CDX2 and MSX1 in NF2 knockout cells reflect a necessary role for NF2 in removing regulatory roadblocks. Furthermore, we found that NF2 and AMOT (angiomotin) cooperatively bind to YAP (yes-associated protein) during mesendoderm formation, thereby preventing YAP activation, independent of canonical MST (mammalian sterile 20-like serine-threonine protein kinase)-LATS (large tumor suppressor serine-threonine protein kinase) signaling. Mechanistically, cardiomyocyte lineage identity was rescued by wild-type and NF2 serine-518 phosphomutants, but not NF2 FERM (ezrin-radixin-meosin homology protein) domain blue-box mutants, demonstrating that the critical FERM domain-dependent formation of the AMOT-NF2-YAP scaffold complex at the adherens junction is required for early cardiomyocyte lineage differentiation. CONCLUSIONS: These results provide mechanistic insight into the essential role of NF2 during early epithelial-mesenchymal transition by sequestering the repressive effect of YAP and relieving regulatory roadblocks en route to cardiomyocytes.

3. TRIM35: A Proposed Gateway to p53-Induced Heart Failure Pathogenesis

   C. J. M. Lee ,  and  R. S. Foo

   2024

   Lee, Chang Jie Mick Foo, Roger S-Y eng Comment Editorial 2024/07/04 Circ Res. 2024 Jul 5;135(2):314-316. doi: 10.1161/CIRCRESAHA.124.324792. Epub 2024 Jul 4.
4. Asparagine Synthetase Marks a Distinct Dependency Threshold for Cardiomyocyte Dedifferentiation

   Y. Zhu ,  M. Ackers-Johnson ,  M. K. Shanmugam ,  L. S. Pakkiri ,  C. L. Drum ,  Y. Chen ,  J. Kim ,  W. G. Paltzer ,  A. I. Mahmoud ,  W. L. Wen Tan ,  C. J. M. Lee ,  J. Jiang ,  D. A. T. Luu ,  S. L. Ng ,  P. Y. Q. Li ,  A. Wang ,  R. Qi ,  G. J. X. Ong ,  T. Ng Yu ,  J. J. Haigh ,  Z. Tiang ,  A. M. Richards ,  and  R. Foo

   2024

   Zhu, Yike Ackers-Johnson, Matthew Shanmugam, Muthu K Pakkiri, Leroy Sivappiragasam Drum, Chester Lee Chen, Yanpu Kim, Johnny Paltzer, Wyatt G Mahmoud, Ahmed I Wen Tan, Wilson Lek Lee, Chang Jie Mick Jiang, Jianming Luu, Danh Anh Tuan Ng, Shi Ling Li, Peter Yi Qing Wang, Anhui Qi, Rong Ong, Gabriel Jing Xiang Ng Yu, Timothy Haigh, Jody J Tiang, Zenia Richards, A Mark Foo, Roger eng T32 HL007936/HL/NHLBI NIH HHS/ 2024/04/08 Circulation. 2024 Apr 8. doi: 10.1161/CIRCULATIONAHA.123.063965.

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   BACKGROUND: Adult mammalian cardiomyocytes have limited proliferative capacity, but in specifically induced contexts they traverse through cell-cycle reentry, offering the potential for heart regeneration. Endogenous cardiomyocyte proliferation is preceded by cardiomyocyte dedifferentiation (CMDD), wherein adult cardiomyocytes revert to a less matured state that is distinct from the classical myocardial fetal stress gene response associated with heart failure. However, very little is known about CMDD as a defined cardiomyocyte cell state in transition. METHODS: Here, we leveraged 2 models of in vitro cultured adult mouse cardiomyocytes and in vivo adeno-associated virus serotype 9 cardiomyocyte-targeted delivery of reprogramming factors (Oct4, Sox2, Klf4, and Myc) in adult mice to study CMDD. We profiled their transcriptomes using RNA sequencing, in combination with multiple published data sets, with the aim of identifying a common denominator for tracking CMDD. RESULTS: RNA sequencing and integrated analysis identified Asparagine Synthetase (Asns) as a unique molecular marker gene well correlated with CMDD, required for increased asparagine and also for distinct fluxes in other amino acids. Although Asns overexpression in Oct4, Sox2, Klf4, and Myc cardiomyocytes augmented hallmarks of CMDD, Asns deficiency led to defective regeneration in the neonatal mouse myocardial infarction model, increased cell death of cultured adult cardiomyocytes, and reduced cell cycle in Oct4, Sox2, Klf4, and Myc cardiomyocytes, at least in part through disrupting the mammalian target of rapamycin complex 1 pathway. CONCLUSIONS: We discovered a novel gene Asns as both a molecular marker and an essential mediator, marking a distinct threshold that appears in common for at least 4 models of CMDD, and revealing an Asns/mammalian target of rapamycin complex 1 axis dependency for dedifferentiating cardiomyocytes. Further study will be needed to extrapolate and assess its relevance to other cell state transitions as well as in heart regeneration.

5. Ultra-Tough Graphene Oxide/DNA 2D Hydrogel with Intrinsic Sensing and Actuation Functions

   S. Chen ,  C. J. M. Lee ,  G. S. X. Tan ,  P. R. Ng ,  P. Zhang ,  J. Zhao ,  K. S. Novoselov ,  and  D. V. Andreeva

   2024

   Chen, Siyu Lee, Chang Jie Mick Tan, Gladys Shi Xuan Ng, Pei Rou Zhang, Pengxiang Zhao, Jinpei Novoselov, Kostya S Andreeva, Daria V eng EDUN C-33-18-279-V12 I-FIM/Ministry of Education (Singapore) through the Research Centre of Excellence program/ Germany 2024/08/05 Macromol Rapid Commun. 2024 Aug 5:e2400518. doi: 10.1002/marc.202400518.

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   Hydrogel devices with mechanical toughness and tunable functionalities are highly desirable for practical long-term applications such as sensing and actuation elements for soft robotics. However, existing hydrogels have poor mechanical properties, slow rates of response, and low functionality. In this work, two-dimensional hydrogel actuators are proposed and formed on the self-assembly of graphene oxide (GO) and deoxynucleic acid (DNA). The self-assembly process is driven by the GO-induced transition of double stranded DNA (dsDNA) into single stranded DNA (ssDNA). Thus, the hydrogel’s structural unit consists of two layers of GO covered by ssDNA and a layer of dsDNA in between. Such heterogeneous architectures stabilized by multiple hydrogen bondings have Young’s modulus of up to 10 GPa and rapid swelling rates of 4.0 x 10(-3) to 1.1 x 10(-2) s(-1), which surpasses most types of conventional hydrogels. It is demonstrated that the GO/DNA hydrogel actuators leverage the unique properties of these two materials, making them excellent candidates for various applications requiring sensing and actuation functions, such as artificial skin, wearable electronics, bioelectronics, and drug delivery systems.

2023

1. Systematic in vivo candidate evaluation uncovers therapeutic targets for LMNA dilated cardiomyopathy and risk of Lamin A toxicity

   C. Y. Tan ,  P. S. Chan ,  H. Tan ,  S. W. Tan ,  C. J. M. Lee ,  J. W. Wang ,  S. Ye ,  H. Werner ,  Y. J. Loh ,  Y. L. Lee ,  M. Ackers-Johnson ,  R. S. Y. Foo ,  and  J. Jiang

   2023

   Tan, Chia Yee Chan, Pui Shi Tan, Hansen Tan, Sung Wei Lee, Chang Jie Mick Wang, Jiong-Wei Ye, Shu Werner, Hendrikje Loh, Ying Jie Lee, Yin Loon Ackers-Johnson, Matthew Foo, Roger S Y Jiang, Jianming eng Research Support, Non-U.S. Gov’t England 2023/10/16 J Transl Med. 2023 Oct 16;21(1):690. doi: 10.1186/s12967-023-04542-4.

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   BACKGROUND: Dilated cardiomyopathy (DCM) is a severe, non-ischemic heart disease which ultimately results in heart failure (HF). Decades of research on DCM have revealed diverse aetiologies. Among them, familial DCM is the major form of DCM, with pathogenic variants in LMNA being the second most common form of autosomal dominant DCM. LMNA DCM is a multifactorial and complex disease with no specific treatment thus far. Many studies have demonstrated that perturbing candidates related to various dysregulated pathways ameliorate LMNA DCM. However, it is unknown whether these candidates could serve as potential therapeutic targets especially in long term efficacy. METHODS: We evaluated 14 potential candidates including Lmna gene products (Lamin A and Lamin C), key signaling pathways (Tgfbeta/Smad, mTor and Fgf/Mapk), calcium handling, proliferation regulators and modifiers of LINC complex function in a cardiac specific Lmna DCM model. Positive candidates for improved cardiac function were further assessed by survival analysis. Suppressive roles and mechanisms of these candidates in ameliorating Lmna DCM were dissected by comparing marker gene expression, Tgfbeta signaling pathway activation, fibrosis, inflammation, proliferation and DNA damage. Furthermore, transcriptome profiling compared the differences between Lamin A and Lamin C treatment. RESULTS: Cardiac function was restored by several positive candidates (Smad3, Yy1, Bmp7, Ctgf, aYAP1, Sun1, Lamin A, and Lamin C), which significantly correlated with suppression of HF/fibrosis marker expression and cardiac fibrosis in Lmna DCM. Lamin C or Sun1 shRNA administration achieved consistent, prolonged survival which highly correlated with reduced heart inflammation and DNA damage. Importantly, Lamin A treatment improved but could not reproduce long term survival, and Lamin A administration to healthy hearts itself induced DCM. Mechanistically, we identified this lapse as caused by a dose-dependent toxicity of Lamin A, which was independent from its maturation. CONCLUSIONS: In vivo candidate evaluation revealed that supplementation of Lamin C or knockdown of Sun1 significantly suppressed Lmna DCM and achieve prolonged survival. Conversely, Lamin A supplementation did not rescue long term survival and may impart detrimental cardiotoxicity risk. This study highlights a potential of advancing Lamin C and Sun1 as therapeutic targets for the treatment of LMNA DCM.

2022

1. Genomic enhancers in cardiac development and disease

   C. G. Anene-Nzelu ,  C. J. M. Lee ,  W. L. W. Tan ,  A. Dashi ,  and  R. S. Y. Foo

   2022

   Anene-Nzelu, Chukwuemeka G Lee, Mick C J Tan, Wilson L W Dashi, Albert Foo, Roger S Y eng Review England 2021/08/13 Nat Rev Cardiol. 2022 Jan;19(1):7-25. doi: 10.1038/s41569-021-00597-2. Epub 2021 Aug 11.

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   The Human Genome Project marked a major milestone in the scientific community as it unravelled the  3 billion bases that are central to crucial aspects of human life. Despite this achievement, it only scratched the surface of understanding how each nucleotide matters, both individually and as part of a larger unit. Beyond the coding genome, which comprises only  2% of the whole genome, scientists have realized that large portions of the genome, not known to code for any protein, were crucial for regulating the coding genes. These large portions of the genome comprise the ’non-coding genome’. The history of gene regulation mediated by proteins that bind to the regulatory non-coding genome dates back many decades to the 1960s. However, the original definition of ’enhancers’ was first used in the early 1980s. In this Review, we summarize benchmark studies that have mapped the role of cardiac enhancers in disease and development. We highlight instances in which enhancer-localized genetic variants explain the missing link to cardiac pathogenesis. Finally, we inspire readers to consider the next phase of exploring enhancer-based gene therapy for cardiovascular disease.

2. The genetic interactions between non-alcoholic fatty liver disease and cardiovascular diseases

   N. W. S. Chew ,  B. Chong ,  C. H. Ng ,  G. Kong ,  Y. H. Chin ,  W. Xiao ,  C. J. M. Lee ,  Y. Y. Dan ,  M. D. Muthiah ,  and  R. Foo

   2022

   Chew, Nicholas W S Chong, Bryan Ng, Cheng Han Kong, Gwyneth Chin, Yip Han Xiao, Wang Lee, Mick Dan, Yock Young Muthiah, Mark D Foo, Roger eng Review Switzerland 2022/08/30 Front Genet. 2022 Aug 10;13:971484. doi: 10.3389/fgene.2022.971484. eCollection 2022.

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   The ongoing debate on whether non-alcoholic fatty liver disease (NAFLD) is an active contributor or an innocent bystander in the development of cardiovascular disease (CVD) has sparked interests in understanding the common mediators between the two biologically distinct entities. This comprehensive review identifies and curates genetic studies of NAFLD overlapping with CVD, and describes the colinear as well as opposing correlations between genetic associations for the two diseases. Here, CVD described in relation to NAFLD are coronary artery disease, cardiomyopathy and atrial fibrillation. Unique findings of this review included certain NAFLD susceptibility genes that possessed cardioprotective properties. Moreover, the complex interactions of genetic and environmental risk factors shed light on the disparity in genetic influence on NAFLD and its incident CVD. This serves to unravel NAFLD-mediated pathways in order to reduce CVD events, and helps identify targeted treatment strategies, develop polygenic risk scores to improve risk prediction and personalise disease prevention.

3. BNIP-2 Activation of Cellular Contractility Inactivates YAP for H9c2 Cardiomyoblast Differentiation

   D. C. P. Wong ,  J. Xiao ,  T. W. Chew ,  M. Pan ,  C. J. M. Lee ,  J. W. Ang ,  I. Yow ,  T. Thivakar ,  M. Ackers-Johnson ,  N. J. W. Lee ,  R. S. Foo ,  P. Kanchanawong ,  and  B. C. Low

   2022

   Wong, Darren Chen Pei Xiao, Jingwei Chew, Ti Weng Pan, Meng Lee, Chang Jie Mick Ang, Jing Wen Yow, Ivan Thivakar, T Ackers-Johnson, Matthew Lee, Nicole Jia Wen Foo, Roger Sik-Yin Kanchanawong, Pakorn Low, Boon Chuan eng Mechanobiology Institute Singapore (MBI)/ National Research Foundation and the Ministry of Education Singapore/ MOE2016-T3-1-002/Ministry of Education (Singapore) Academic Research Fund Tier 3/ MOE2019-T2-2-014/Ministry of Education (Singapore) Academic Research Fund Tier2/ Research Support, Non-U.S. Gov’t Germany 2022/08/18 Adv Sci (Weinh). 2022 Nov;9(31):e2202834. doi: 10.1002/advs.202202834. Epub 2022 Aug 17.

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   Rho GTPases and Hippo kinases are key regulators of cardiomyoblast differentiation. However, how these signaling axes are coordinated spatiotemporally remains unclear. Here, the central and multifaceted roles of the BCH domain containing protein, BNIP-2, in orchestrating the expression of two key cardiac genes (cardiac troponin T [cTnT] and cardiac myosin light chain [Myl2]) in H9c2 and human embryonic stem cell-derived cardiomyocytes are delineated. This study shows that BNIP-2 mRNA and protein expression increase with the onset of cTnT and Myl2 and promote the alignment of H9c2 cardiomyocytes. Mechanistically, BNIP-2 is required for the inactivation of YAP through YAP phosphorylation and its cytosolic retention. Turbo-ID proximity labeling corroborated by super-resolution analyses and biochemical pulldown data reveals a scaffolding role of BNIP-2 for LATS1 to phosphorylate and inactivate YAP in a process that requires BNIP-2 activation of cellular contractility. The findings identify BNIP-2 as a pivotal signaling scaffold that spatiotemporally integrates RhoA/Myosin II and LATS1/YAP mechanotransduction signaling to drive cardiomyoblast differentiation, by switching the genetic programming from YAP-dependent growth to YAP-silenced differentiation. These findings offer insights into the importance of scaffolding proteins in bridging the gap between mechanical and biochemical signals in cell growth and differentiation and the prospects in translational applications.

4. CTCF mediates the Activity-by-contact derived cis-regulatory hubs

   C. J. M. Lee ,  Loı̈c Mangnier ,  Cory C. Padilla ,  Dominic Paul Lee ,  Wilson Tan ,  Peter Yiqing Li ,  Wen Hao Zheng ,  Louis Gan ,  Ching Kit Chen ,  Yee Phong Lim ,  Rina Miao Qin Wang ,  Steve Bilodeau ,  Alexandre Bureau ,  Roger Sik-Yin Foo^ ,  and  Chukwuemeka George Anene-Nzelu^

   bioRxiv, 2022

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   The 3D chromatin architecture establishes a complex network of genes and regulatory elements necessary for transcriptomic regulation in development and disease. This network can be modeled by cis-regulatory hubs (CRH) which underscore the local functional interactions between enhancers and promoter regions and differ from other higher order chromatin structures such as topological associated domains (TAD). The Activity-by-contact (ABC) model of enhancer-promoter regulation has been recently used in the identification of these CRHs, but little is known about the role of CTCF on the ABC scores and the consequent impact on CRHs. Here we show that the loss of CTCF leads to a reorganization of the enhancer-promoter interactions resulting in a re-distribution of ABC scores of the putative enhancers in C57/bl6 mouse hearts. The loss of CTCF also leads to a global reduction of the total number of CRHs, and an increase in the size of the CRHs due to increase in the number of elements within each hub. In addition, CTCF loss led to more CRHs that cross TAD boundaries. These results provide another layer of evidence to support the importance of CTCF in the formation of regulatory networks necessary for gene regulation.Competing Interest StatementThe authors have declared no competing interest.ChIP-seqChromatin immunoprecipitation with sequencingATAC-seqAssay for Transposase Accessible Chromatin with sequencingHi-CHigh throughput chromatin conformation capture with sequencingABCActivity by contact algorithm

2020

1. Assigning Distal Genomic Enhancers to Cardiac Disease-Causing Genes

   *C. G. Anene-Nzelu ,  *W. L. W. Tan ,  *C. J. M. Lee ,  Z. Wenhao ,  A. Perrin ,  A. Dashi ,  Z. Tiang ,  M. I. Autio ,  B. Lim ,  E. Wong ,  H. S. Tan ,  B. Pan ,  M. P. Morley ,  K. B. Margulies ,  T. P. Cappola ,  and  R. S. Foo

   2020

   1524-4539 R01 HL105993/HL/NHLBI NIH HHS/United States Letter United States 2020/09/01 Circulation. 2020 Sep;142(9):910-912. doi: 10.1161/CIRCULATIONAHA.120.046040. Epub 2020 Aug 31.

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2. Epigenomes of Human Hearts Reveal New Genetic Variants Relevant for Cardiac Disease and Phenotype

   W. L. W. Tan ,  C. G. Anene-Nzelu ,  E. Wong ,  C. J. M. Lee ,  H. S. Tan ,  S. J. Tang ,  A. Perrin ,  K. X. Wu ,  W. Zheng ,  R. J. Ashburn ,  B. Pan ,  M. Y. Lee ,  M. I. Autio ,  M. P. Morley ,  W. L. Tam ,  C. Cheung ,  K. B. Margulies ,  L. Chen ,  T. P. Cappola ,  M. Loh ,  J. Chambers ,  S. Prabhakar ,  R. S. Y. Foo ,  and  Charge-EchoGen Consortium Charge-Heart Failure Working Group

   2020

   Tan, Wilson Lek Wen Anene-Nzelu, Chukwuemeka George Wong, Eleanor Lee, Chang Jie Mick Tan, Hui San Tang, Sze Jing Perrin, Arnaud Wu, Kan Xing Zheng, Wenhao Ashburn, Robert John Pan, Bangfen Lee, May Yin Autio, Matias Ilmari Morley, Michael P Tam, Wai Leong Cheung, Christine Margulies, Kenneth B Chen, Leilei Cappola, Thomas P Loh, Marie Chambers, John Prabhakar, Shyam Foo, Roger S Y eng R01 HL105993/HL/NHLBI NIH HHS/ Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov’t 2020/06/13 Circ Res. 2020 Aug 28;127(6):761-777. doi: 10.1161/CIRCRESAHA.120.317254. Epub 2020 Jun 12.

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   RATIONALE: Identifying genetic markers for heterogeneous complex diseases such as heart failure is challenging and requires prohibitively large cohort sizes in genome-wide association studies to meet the stringent threshold of genome-wide statistical significance. On the other hand, chromatin quantitative trait loci, elucidated by direct epigenetic profiling of specific human tissues, may contribute toward prioritizing subthreshold variants for disease association. OBJECTIVE: Here, we captured noncoding genetic variants by performing epigenetic profiling for enhancer H3K27ac chromatin immunoprecipitation followed by sequencing in 70 human control and end-stage failing hearts. METHODS AND RESULTS: We have mapped a comprehensive catalog of 47 321 putative human heart enhancers and promoters. Three thousand eight hundred ninety-seven differential acetylation peaks (FDR [false discovery rate], 5%) pointed to pathways altered in heart failure. To identify cardiac histone acetylation quantitative trait loci (haQTLs), we regressed out confounding factors including heart failure disease status and used the G-SCI (Genotype-independent Signal Correlation and Imbalance) test(1) to call out 1680 haQTLs (FDR, 10%). RNA sequencing performed on the same heart samples proved a subset of haQTLs to have significant association also to gene expression (expression quantitative trait loci), either in cis (180) or through long-range interactions (81), identified by Hi-C (high-throughput chromatin conformation assay) and HiChIP (high-throughput protein centric chromatin) performed on a subset of hearts. Furthermore, a concordant relationship between the gain or disruption of TF (transcription factor)-binding motifs, inferred from alternative alleles at the haQTLs, implied a surprising direct association between these specific TF and local histone acetylation in human hearts. Finally, 62 unique loci were identified by colocalization of haQTLs with the subthreshold loci of heart-related genome-wide association studies datasets. CONCLUSIONS: Disease and phenotype association for 62 unique loci are now implicated. These loci may indeed mediate their effect through modification of enhancer H3K27 acetylation enrichment and their corresponding gene expression differences (bioRxiv: https://doi.org/10.1101/536763). Graphical Abstract: A graphical abstract is available for this article.

2019

1. Robust CTCF-Based Chromatin Architecture Underpins Epigenetic Changes in the Heart Failure Stress-Gene Response

   D. P. Lee ,  W. L. W. Tan ,  C. G. Anene-Nzelu ,  C. J. M. Lee ,  P. Y. Li ,  T. D. A. Luu ,  C. X. Chan ,  Z. Tiang ,  S. L. Ng ,  X. Huang ,  M. Efthymios ,  M. I. Autio ,  J. Jiang ,  M. J. Fullwood ,  S. Prabhakar ,  E. Lieberman Aiden ,  and  R. S. Foo

   2019

   Lee, Dominic Paul Tan, Wilson Lek Wen Anene-Nzelu, Chukwuemeka George Lee, Chang Jie Mick Li, Peter Yiqing Luu, Tuan Danh Anh Chan, Cheryl Xueli Tiang, Zenia Ng, Shi Ling Huang, Xingfan Efthymios, Motakis Autio, Matias I Jiang, Jianming Fullwood, Melissa Jane Prabhakar, Shyam Lieberman Aiden, Erez Foo, Roger Sik-Yin eng Research Support, Non-U.S. Gov’t 2019/02/06 Circulation. 2019 Apr 16;139(16):1937-1956. doi: 10.1161/CIRCULATIONAHA.118.036726.

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   BACKGROUND: The human genome folds in 3 dimensions to form thousands of chromatin loops inside the nucleus, encasing genes and cis-regulatory elements for accurate gene expression control. Physical tethers of loops are anchored by the DNA-binding protein CTCF and the cohesin ring complex. Because heart failure is characterized by hallmark gene expression changes, it was recently reported that substantial CTCF-related chromatin reorganization underpins the myocardial stress-gene response, paralleled by chromatin domain boundary changes observed in CTCF knockout. METHODS: We undertook an independent and orthogonal analysis of chromatin organization with mouse pressure-overload model of myocardial stress (transverse aortic constriction) and cardiomyocyte-specific knockout of Ctcf. We also downloaded published data sets of similar cardiac mouse models and subjected them to independent reanalysis. RESULTS: We found that the cardiomyocyte chromatin architecture remains broadly stable in transverse aortic constriction hearts, whereas Ctcf knockout resulted in approximately 99% abolition of global chromatin loops. Disease gene expression changes correlated instead with differential histone H3K27-acetylation enrichment at their respective proximal and distal interacting genomic enhancers confined within these static chromatin structures. Moreover, coregulated genes were mapped out as interconnected gene sets on the basis of their multigene 3D interactions. CONCLUSIONS: This work reveals a more stable genome-wide chromatin framework than previously described. Myocardial stress-gene transcription responds instead through H3K27-acetylation enhancer enrichment dynamics and gene networks of coregulation. Robust and intact CTCF looping is required for the induction of a rapid and accurate stress response.